P458: TRANSCRIPTIONAL AND TRANSLATIONAL SIGNATURES OF TRIPLE MUTATED DNMT3AKO/FLT3ITD/NPM1C MICE SHOW ALTERED RNAS PROCESSING AND CELL CYCLE PROGRESSION
Authors: A. Scialdone, D. Sorcini, A. Stella, V. Tini, A. Marra, C. Rompietti, F. De Falco, E. Dorillo, F. M. Adamo, E. C. Barcelos, R. Arcaleni, M. P. Martelli, P. Sportoletti°, B. Falini°;° co-last author
Journal: HemaSphere (2022)

Background: The top ranking recurrently mutated genes in AML are NPM1, FLT3 and DNMT3A1,2. Co-occurrence of these three mutations is associated to a unique AML subset characterized by high peripheral blood and bone marrow blast counts, normal cytogenetic and very poor EFS and OS3. Nevertheless, the cooperative leukemogenic mechanisms of these three mutations have not been deeply explored and a successful targeting treatment of AML patientsDNMT3Amut/FLT3-ITD/NPM1c still represents an unmet clinical need. Aims: To dissect how DNMT3A loss of function, FLT3 Internal Tandem Duplication (ITD) and NPM1 mutations (NPM1c) cooperate in driving AML onset and progression through a picture of the transcriptome and the translatome of triple mutated AML cells. Methods: Mx1-Cre-NPM1c and/or Mx1-Cre-DNMT3AKO and FLT3ITD mutant mice were crossed to obtain the following lines: DNMT3AKO/FLT3ITD/NPM1c, FLT3ITD/DNMT3AKO and FLT3ITD/NPM1c. Peripheral blood (PB) was periodically taken and analysed, previous anesthesia, while bone marrow (BM) and spleen were harvested two months after pIpC induction or at conclamate disease (n=7 each group). To investigate the mechanisms underlying the triple mutated mice phenotype, we looked BM leukemic samples from all groups both at transcriptional level by RNA sequencing (RNAseq) and at translational level (protein synthesis capacity of AML blasts) by Ribosome footprinting (Riboseq)4. Unique Differentially Expressed Genes (DEGs) (filtered by p-value<0.05 and FC >1.5) belonging to FLT3ITD/DNMT3AKO/NPM1cvs FLT3ITD/DNMT3AKO were identified by Venn diagram and used in enrichment analysis on Reactome database and MSigDB (C6-oncogenic signature). Results: The FLT3ITD/DNMT3AKO/NPM1c, DNMT3AKO/FLT3ITD and FLT3ITD/NPM1c displayed a fully penetrant leukemic phenotype. As expected, FLT3ITD/ DNMT3AKO/NPM1c showed a more aggressive disease characterized by higher WBC counts, more pronounced blasts infiltration of the spleen and shorter survival (>2 fold vs double mutants). The FLT3ITD/DNMT3AKO/NPM1c mice displayed 1429 (842 UP and 587 DOWN) DEGs compared to FLT3ITD/DNMT3AKO mice. The enrichment analysis suggested that DNMT3A and NPM1 mutations, in a constitutive FLT3ITD expression context, cooperate to activate transcriptional programs sustaining the proliferation and maintenance of blasts through concerted upregulation of a number of pathways, including the Anaphase-Promoting Complex (APC), which regulates cell cycle progression, the m-TOR-mediated autophagy inhibition, the Rho-GTPases effectors (with Rac1 overexpression), the Hippo-mediated self renewal, the HOXA9 signature and TP53-regulated apoptosis. Interestingly, genes involved in transport of mature transcripts to cytoplasm and nuclear RNAs metabolism were also upregulated. In keeping with these RNAseq findings, the Riboseq data showed that both the quote of processed transcripts (15 vs 9%) and the percentage of processed pseudogenes were more abundant in triple vs double mutated mice. On the other hand, the long intergenic noncoding RNAs (lincRNA) fraction was reduced in triple mutants. This finding may reflect dysfunctional remodeling of tumor microenvironment, contributing to AML cells immune escape in the BM5. Image:Summary/Conclusion: The leukemic DNMT3AKO/FLT3ITD/NPM1c genotype is characterized by unique transcriptional and translational signatures. The information about deregulated pathways and altered protein synthesis establishes the rational basis for designing new therapeutic approaches in this poor prognosis AML genotype.